Interference control panel for evaluation of analytical assays for samples derived from blood

ABSTRACT

The invention relates to quality control of analytical assays, particularly NAT assays of blood samples containing nucleic acids. A control panel containing quantified amounts of substances known to interfere with an analytical assay is used and compared with a reference sample in the analytical assay. A comparison of the assay results interference panel validates the assay and can serve as a periodic quality control check for the analytical assay as well as related methods and protocols. The use of the control panel of the invention can also determine whether interfering substances are present and establish under what conditions the analytical assay reliable.

BACKGROUND OF THE INVENTION

Blood samples are extensively used for clinical diagnosis and in medicalresearch. In many assay formats, the presence of certain components inblood can interfere with the assay and render the test resultsunreliable or unusable. Interference is typically manifested as aninhibition of chemical reactions in the assay that reduces theperformance and compromises the integrity of the assay and its result.

Patient samples may be compromised in a clinical setting by conditionssuch as poor handling, haemolysis, icterus, or lipemia. It is forexample well known that HIV patients, treated with protease inhibitors,often show increased triglyceride levels. Yet another example of acompromised sample is cadaveric samples, which are often tested beforeorgan transplantation. Cadaveric samples can be very challenging fornucleic acid tests, due to their potentially inhibitory nature fromlysed or degraded tissues.

Blood samples, or samples derived from whole blood, are often analyzedfor a nucleic acid analyte by the Polymerase Chain Reaction (PCR), otherNucleic Acid Amplification Technologies (NAT), or other nucleic aciddetection technologies. NAT and PCR-based diagnosis of disease,infections, and genetic variations, as well as forensic analysis andblood typing are well known. It is also known that contaminants or PCRinhibitory substances such as lipids, hemoglobin, bilirubin orfrequently administered drugs and anticoagulants can interfere with thePCR assay.

PCR-based assays rely on amplification and detection of nucleic acidspresent in the blood samples. These reactions can be dramaticallyreduced or blocked by the presence of contaminants or natural componentsof blood that inhibit chemical or biochemical reactions that occur inthe assay. Blood components known as PCR inhibitors includeimmunoglobulin, heme, hemoglobin, leukocyte DNA, and common bloodadditive such as the anticoagulant heparin. Therefore, the usefulness ofPCR-based detection of microorganisms, pathogens and other targets incomplex biological samples, such as clinical, environmental, and foodsamples, is limited in part by the presence of substances that inhibitthe fundamental amplification reaction at PCR or which reduce theamplification efficiency.

Because the potential for contamination and interference in PCR-basedassays is well known, a variety of different approaches have beenstudied to attempt to prevent the inhibition. In one approach, theinhibition caused by specific substances is tested to attempt tocompensate for their presence in a test sample or assay.

Solutions that samples used for assays typically are, or may be,converted into a liquid form containing compounds that inhibit chemicalor biochemical reactions in an assay. The agents that cause interferenceor inhibition in the inhibitory testing assay include: hemoglobin,L-ascorbic acid, free fatty acids, iron, heme, triglycerides, drugs,bilirubin, conjugated bilirubin, bicarbonate, pH extremes, proteins,bile acids, larger amounts of DNA, or keto-acids. These inhibitors mayinterfere with cell lysis, degrade or capture of the nucleic acids,inactivate Taq polymerase or degrade the specificity of this enzyme, orotherwise interfere with enzymes used in nucleic acid amplification ordetection technologies. In particular reverse transcription PCR(rt-PCR), which initially reverse transcribes RNA into cDNA, is verysensitive to the presence of inhibitors. In an attempt to preserve thefidelity of the assay, different methods of sample preparation have beendeveloped to remove the inhibitory effect of these blood-derivedcomponents.

It is possible to attempt to control for inhibitory substances bymonitoring the presence or absence of PCR product(s) at the end ofthermal cycling by gel electophoresis, dot blots, high-pressure liquidchromatography or microtiter or plate-based, calorimetric assay. Thequantitative effect of inhibitors on DNA synthesis can also be studiedby measuring the efficiency of incorporation of radiolabelednucleotides. Recently, thermal cyclers with real-time detection of PCRproduct accumulation were introduced, offering a new possibility tostudy amplification efficiency and/or DNA synthesis efficiency. Mostcommonly a known amount of an internal control molecule, which shouldbehave similar to the target, is added into each PCR reactions. A changefrom the expected signal generated by the internal control can indicatethe presence of inhibitors but can also change if the assay was notperformed correctly. An internal control used for quantification issometimes referred to as Quantification Standard (QS).

A resolution of the problem of inhibiting substances in assays can beattempted by sample preparation techniques. PCR-inhibitory componentsinclude salts, complex polysaccharides, heme protein in blood, RNases,DNases, feces, some detergents (e.g., SDS), DNA intercalating substances(e.g., intercalating dyes), humic substances in soil, melanin, collagen,myoglobin, alcohol, calcium ions, lactoferrin, proteases, proteinases inmilk, and urea in urine. Significant effort is being devoted to thedevelopment of sample preparation methods to remove these substances andovercome the problems of inhibition in the reactions that occur in anassay. Different processing techniques are also being employed to reducethe effect of inhibitors. For example, aqueous two phase-systems,boiling, density gradient centrifugation, dilution, DNA extractionmethods, enrichment media, filtration, and immunological techniques havebeen used to attempt to avoid the effect of inhibitory substances in PCRanalyses. The thermostable DNA polymerase is perhaps the most importanttarget site of PCR-inhibiting substances. The most widely usedpolymerase in PCR-based methods for the detection of microorganisms isTaq DNA polymerase from Thermus aquaticus. Other DNA polymerases withmanganese instead of magnesium as cofactor are commonly used for rt-PCR.Other systems use a reverse transcriptase in combination with a DNApolymerase for rtPCR. Taq DNA polymerase, as well as many other PCRenzymes, can be degraded by proteinases, denatured by phenol ordetergents, and inhibited by blocking of the active site by theinhibitor, which is the effect of the heme protein. Inhibitors can alsowork on the substrate by decaying DNA or RNA. RNases in plasma are knownto destroy RNA, or DNases can destroy DNA.

One approach for quality control of analytical assays is to use asample-processing control as an internal control to verify adequateprocessing of the target analyte. This monitors the presence ofinhibitors to avoid a false negative result, or incorrectquantification. If the system fails with this control, then there is aninvalid result.

While common interfering substances are well known, it remains difficultand inconvenient to prepare constant, quantified controls for analyticalassays. The reliability of standard solutions may be questionable orconcentrations may not be calculated correctly. Also, it istime-consuming to be constantly preparing standards and calibrating themfrom week to week. This is particularly true for clinical samples, wherethere is high volume of testing, yet these are samples which mustconform to rigorous quality control standards. Further, patient samplesmay be contaminated, or be presented in less than optimum condition.Because sample size and availability may be limited, however, it isoften important that these critical samples be accurately analyzed.

Known guidelines recognize two primary limitations of interferencetesting. Properties of compounds added to a serum pool may be differentfrom those of the compound naturally circulating in vivo. Also, thepresence of more than one interfering substance may offset or enhancethe concentrations of interfering substance and analyte tested. Variouscombinations of interfering substances in various combinations,therefore, should be evaluated in an assay.

To assure optimum performance of the analytical assay, a test assayincludes several types of controls in addition to the test sample. Anegative control, usually with a key ingredient missing, indicates apositive test result can be trusted. A positive control, usually a knownamount of the analyte of interest, indicates the assay is functional. Aninternal control has a known substance added to the test sample beforeanalysis, while an external control compares the test sample with othersamples of substances run in the same assay. Because analytical assayscan be sensitive and are designed to detect very small quantities ofanalyte, it is very important to include complementary controls toevaluate an analytical assay for acceptance criteria.

Accepted guidelines for estimation of interference characteristicsrecommend evaluating the effect of potentially interfering substancesadded to the sample of interest, as well as evaluating the bias ofindividual patient specimens in comparison to a highly specificcomparative measurement procedure (Clinical and Laboratory StandardsInstitute. Interference Testing in Clinical Chemistry; ApprovedGuideline—Second Edition, 2005, p. 13). An interference screen can beprepared by adding a potentially interfering substance to a sample pooland evaluating bias relative to a control portion of the same pool,which is termed paired-difference testing. An interference screen, withmany substances at relatively high concentrations, simulatesinterference and provides a standardized evaluation to complement actualpatient samples.

It would be very useful to have calibrated substances prepared in aninterference panel to determine the presence of interfering substancesand to evaluate the performance of an analytical assay. This would alsoallow the standardized comparison of interfering substances on differentassays or diagnostic systems.

SUMMARY OF THE INVENTION

The invention provides a panel of calibrated substances known to havethe potential to interfere with an assay of interest. The assay measuressubstances in plasma or blood-derived samples. A known quantity of ananalyte of interest is added to members of the panel and then tested inthe assay. In a preferred embodiment, the interference panel is used inNucleic Acid Detection assays including a nucleic acid extraction andamplification analytical assay. The analyte of interest may be extractedfrom serum, plasma, whole blood or other blood fraction.

The members of the interference panel are substances chosen to mimicpotential interfering substances or clinical conditions that couldadversely affect the analytical assay. These could include haemolysis,icterus, lipemia, or frequently administered drugs or anticoagulants,such as heparin. The panel is designed to test the limits of the assaysystem to validate the assay thereby confirming that assay results areaccurate and reliable, under conditions in which the sample itself maynot be optimal. The interference panel monitors the effectiveness andefficiency of the extraction, amplification and detection of the targetanalyte in the presence of a defined amount of a potential inhibitor.The panel allows determining which potential inhibitor interferes atwhich level with an assay. This information can be used to setacceptance criteria or warn the user of this assay e.g. in the packageinsert about possible problems in case this inhibitor is present above acertain level. Further the panel can be used to validate, verify orotherwise confirm that an assay system is performing consistent with itsdesign by performing the assay in the presence of a known amount ofinhibitors.

The panel members are provided in standardized vial volumes at knownconcentrations. The analyte of interest is spiked into each panel memberat various known concentrations, and each sample is analyzed within thereportable dynamic range of the assay. The level of interference causedby the interference panel member can be quantified by recording andanalyzing the extent to which the interference affects the performanceof the assay, for example, the delay in threshold cycles of the PCRreaction, or the decrease in quantity of product in the presence of theinterfering substance. The concentration of potential inhibitor can bemodulated by adding the target analyte (e.g. a virus) in differentvolumes. Adding 100 μL of a non-inhibitory liquid containing a certainnumber of viruses into the vial containing the potential inhibitor woulddilute the potential inhibitor more than the same number of virus addedin a 10 μL volume of a non-inhibitory liquid.

The method of the invention includes performing an assay in the presenceof potentially interfering substances and determining the extent ofinterference, typically by comparing the results to evaluate quality andperformance of an assay, to verify or quantify interference effects, orto confirm observed interference in patient samples. The panel of theinvention can be applied to custom design assays, and verification ofassay of off-the-shelf performance. By comparison of the reactions ofthe test sample, the normal plasma sample and the panel members, thepanel can also determine the acceptable levels of interfering substancesfor test results.

The inhibition panel of the present invention contains potentiallyinterfering substances that frequently occur due to clinical conditionssuch as haemolysis, icterus, or lipemia, cadaveric samples, or due toadministered drugs or anticoagulants such as heparin. The level ofinterfering substance in each panel member is within the range oftypically occurring human specimens. The panel may also contain a normalreference matrix (EDTA plasma), which can be used as a baseline forcomparison. The interference panel can also include common blood-borneviruses, such as HCV and HBV, and different internal controls spikedinto each panel member in equal quantities.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of detecting inhibition that may beobserved in analytical assays as the result of interfering substancescommonly found in serum, plasma or whole blood specimens. The membersare designed to mimic the potential interfering inhibitor componentssubstances and clinical conditions, such as haemolysis, icterus, andlipemia, and also contains representative substances that are frequentlyused as drugs or anticoagulants (such as heparin) which may causeinhibition of certain assays.

As an external quality control, the inhibition panel provides a methodto test the detection and sensitivity limits of an analytical assaysystem and determines whether a subject assay can report proper resultsunder conditions in which the sample is not optimal. The inhibitionpanel also verifies the effectiveness of any sample preparation orextraction method and screens for the presence or absence of suspectedinhibitors in an analytical assay or system after sample preparation.Further, it allows a standardized check that internal controls or QS areable to indicate the presence or absence of an inhibitor.

As part of a discrete method for assay evaluation, the inhibition panelevaluates the efficiency of the extraction methodology, to determinethat extraction methods or assay systems functionally eliminate anyspecific inhibitors that may be present with analytes found in wholeblood and plasma.

As part of a discrete method for extraction or system evaluation, theinhibition panel verifies that a methodology utilized in samplepreparation has effectively eliminated the inhibitors or interferingsubstances so that instruments are validated according to ClinicalLaboratory Improvement Amendments (CLIA) regulations.

The inhibition panel and assay of the present invention can alsovalidate or verify the performance of a certain assay technique,platform or kit such that when a sample of known concentration is spikedinto each panel member, panel members are analyzed in parallel and thequality or performance of the assay can be determined. For example, PCRresults are evaluated to determine whether or not the amplificationreaction occurring in each panel member shows any difference from thenon-inhibitory reference control typically used in the assay. Thus,inhibiting or interfering substances that are present in whole blood orplasma are detected by comparison of the separate data generated fromthe reaction that occurs for each panel member and with the referenceplasma sample.

Assays that rely on the binding reaction between an antigen and anantibody are known as immunoassays and are often used for diagnosticpurposes. As used herein, “immunoassay” includes methods which utilizethe antibody and a label to detect an antigen in human body fluids, cellor tissue extracts. Other immunoassay-based diagnostic techniquesinclude competitive binding assays, direct, indirect or double antigensandwich assays (e.g. for the detection of antibodies directed againstinfectious disease pathogens) and immunoprecipitation assays [Zola H.,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)pp. 147-158]. Antibodies used in diagnostic assays are typically labeledwith a detectable moiety, which can be a radioisotope, a fluorescent orchemilluminescent compound used in conjunction with an enzyme-linkedantibody. In the case of double antigen sandwich assays, the antibodybinding antigen is labeled.

Antibodies which specifically bind blood-derived antigens areparticularly useful in assays for the diagnosis of conditions ordiseases characterized by altered expression of proteins in the body.Immunoassays are also used to monitor the progress of disease, detectpregnancy, detect infections and several other important diagnosticgoals. Immunoassays are subject to inhibition by any substance thatinterferes with the highly specific binding event between an antibodyand antigen. The reaction site in an antibody or antigen reaction ishighly sensitive and depends on the affinity of the antibody. Theverification or validation of an assay or the identification of aninhibitory substance is achieved by testing members of the inhibitionpanel in an immunoassay wherein the inhibition panel members are reactedwith a quantity of the antigen and antibody. The extent to which thebinding reaction is inhibited can be detected and analyzed in the samemanner as is described elsewhere herein to correlate the inhibitorysubstance to the reaction between the antibody and the antigen for whichthe antibody is specific and to evaluate the quality and performance ofan immunoassay.

The use of the inhibition panel also permits comparison and evaluationof entire systems, providing the opportunity to evaluate and choose thebest assay protocols or protocol a system. Furthermore, the inhibitionpanel permits the evaluation of the characteristics of a sample sourcesuch that limits of other parameters can be assigned to patient samplesto set any requirements for testing, e.g., a maximal level of hemoglobinor heparin may be set for sample acceptance.

The components of the inhibition panel of invention are preferablyderived from whole blood or blood derivatives. In some panel members,human plasma is combined with an interfering substance at a levelappropriate to mimic naturally occurring patient specimens. Theinhibition components may include different kinds of plasma, serum, orother cell-free derived components of whole blood. The components may bederived from either fresh blood, frozen blood or from cadaveric bloodsamples.

A possible source of cadaveric samples is blood drawn from a cadaverbefore embalmment. Blood of dead animals (e.g. pigs, bovine, othermammals or birds) could possibly serve as an alternative source to humancadaveric blood. Another alternative to cadaveric samples is collectinghuman whole blood and letting it sit for extended period of time tosimulate body decay before formulation of a product. A possible causefor the inhibition of cadaveric samples is the presence of heme and DNA,both released after lysis of blood cells. This scenario can be mimickedby e.g. partially or fully lysing donated whole blood (whole bloodlysate) or mixing plasma with DNA and hemoglobin or heme or iron orlysed red blood cells at physiological levels.

The interfering effects of such substances on general NAT and EIA assayshas been established and documented. It is critical to consistentlymonitor the effect of such interfering substances in the performancecharacteristics of the assay validated by the end user. This isespecially true of Analyte Specific Reagents (ASRs) and homebrew assays,where no data is available to verify the performance of the assay in thepresence of such substances. The use of a standardized interferencepanel allows direct comparison of interference data in differentsettings.

The invention can be used to evaluate any sample preparation platform(extraction step, including sample purification and concentration) andsubsequent analytical assay (such as real-time PCR). The volumes of thefinal samples spiked with the analyte and/or the final concentrations ofthe analyte may be adjusted by the end user so that they are within therecommended levels for the assay of interest. The frequency of the usageis determined by the end user, and will depend upon evaluationrequirements of the individual laboratory.

The components described are formulated to mimic normal human specimenscontaining naturally occurring interfering substances, includinginterfering substances due to medication, or interfering substancesintroduced during routine blood collection processes.

The panel can be designed for use with any assay system used to detect abroad number of analytes derived or stored in whole blood or bloodderivatives. Further, the concentrations of each potentially interferingsubstance used as a member of the panel can be varied, i.e., from low tomedium to high concentrations in panel members to establish a broadrange of testing parameters or to establish discrete levels ofinhibition for testing or comparison. Accordingly, the levels ofinterfering substances present in each panel member can be determined tobe within the range observed in clinical patient specimens that aretested for many different analytes.

Although there is a flexibility in the type of analytes which may beused with the panel, all inhibition components, including thenon-inhibitory reference sample, should be spiked with an equal volume(and concentration) of the desired analyte, so that theinterference/non-interference claims of the extraction and/or assaysystem are consistently evaluated. One or more user definednon-inhibitory reference samples can be used in conjunction with, or canreplace a non-inhibitory reference sample if one is provided with theinhibitory panel members.

The following Examples 1-6 describe preparation of representativeinterference panel members to use in the method of the invention.Examples 7-8 describe the practice of embodiments of the invention wherethe inhibition panel is used to evaluate an assay. As used herein, theterm “evaluation” means to assess the reliability of an assay in thepresence of potentially inhibitory substances including verification ofperformance or accuracy or confirmation of any aspect of assayperformance or reliability. This includes assay validation, verificationof the reliability, consistency, reproducibility, quality, andperformance characteristics of an assay, including non-clinicalperformance studies.

EXAMPLE 1 Manufacture of 10% Hemoglobin Solution

Appropriate safety procedures were followed to avoid transmission ofblood-borne infections. Lyophilized human hemoglobin was dissolved in 1×THE buffer (10 mM Tris, 0.2M NaCl, 1 mM EDTA, pH 7.4) to prepare a 10%solution. Extreme caution was utilized in the handling of lyophilizedhemoglobin because it is electrostatic and easily dispersed. Thesolution was stirred for at least 30 minutes at room temperature, andtested using a spectrophotometer (such as the spectrophotometer soldunder the name HemoCue®, or the equivalent).

EXAMPLE 2 Manufacture of 1%, 2% and 4% Hemolyzed Plasma Panel Members

The dilution factor was calculated for the volume of hemolyzed plasmarequired. The appropriate amount of 10% hemoglobin was measured to give1%, 2% or 4% concentration of hemoglobin. The volume of NAT-DM EDTAdiluent was calculated. Both diluents and analyte stocks were measuredgravimetrically, assuming a density of 1.0 g/ml. The desired volume ofdiluent was added to a container, and the desired volume of 10%hemoglobin was added. A lid was placed on the container, and thesolution stirred slowly for 30 minutes or until completely mixed at roomtemperature. The solution was tested with a spectrophotometer (HemoCue®)to verify the hemoglobin concentration was within 10% of the desiredconcentration. The solution was stored at 2-8 degrees C. and used withinone week of preparation.

EXAMPLE 3 Manufacture of Concentrated Fatty Plasma (Cold Fat Depletion)

Concentrated fatty plasma may be prepared by extraction of triglyceridesfrom cold fat by the following process:

A water bath was filled with distilled H₂O, then adjusted to 37° C. Bagsof cold fat were allowed to thaw for 30 minutes in the 37° C. waterbath. Once the plasma bags were thawed and no apparent cryoprecipitateare visible, the plasma bags were placed on a bench toweled flat surfaceto absorb excess moisture. The plasma bags were selected based on thecoloration. The plasma bags were inspected and placed on white paperthen visually evaluated by assessing the clarity and the obscurity ofthe plasma bag under the follows criteria:

i. Pale green to dark yellow plasma is high in fat ii. Light yellow towhite plasma is above average fat iii. Orange-yellowish gold plasma haslow fat content

Four plasma bags of 300 mL fill volume each were chosen and hung hookscustomized for plasma bag holes. Using a tubing connected with a needleor pointed syringe adapter, the septum opening was cut and a tube wasinserted that is attached by a needle inside the bag opening.

300 mL centrifuge containers were filled with plasma from the bags byattaching the end of the tube running from the plasma bag into thecentrifuge container, and without overfilling, the centrifuge tube wasfilled to about 255 mL. The 4 plasma bags permitted about five 300 mLcentrifuge containers to be filled to about 255 mL per container. Afterthe volume of plasma was collected, it was placed in a 4° C.refrigerator for 2 hours to cool. The containers were centrifuged at20,000×g (RCF) at a temperature setting of 4° C. for an hour. The liquidphase (supernatant) from the opposite side of the container from wherethe fat bodies have adhered was carefully decanted and the fat wascarefully pipetted out. At this point of the process, some of the fatadhered to the side of the container as well as floated to the top ofthe supernatant, due to the low density of the fat to plasma liquiddensity.

A second ultracentrifugation was performed at 20,000×g (RCF) at atemperature setting of 4° C. for an hour. The liquid (supernatant) wasdecanted and carefully pipetted to remove the fat as above. Thesupernatant was started in 2-8° C. refrigerator overnight. The followingday, if there was further separation, the residual supernatant wasdecanted to transfer the fat to a container and placed in short-termstorage at 2-8° C. To save the remaining liquid (supernatant) of theplasma, the residual supernatant was kept at 2-8° C., or the fat wasconcentrated by performing the process as described above. The level oftriglycerides in this concentrated fatty plasma was measured. Lipemicplasma was diluted with normal human plasma so that the finalconcentration of triglycerides was 3000 mg/dL.

EXAMPLE 4 Manufacture of Lipemic Human Plasma

The volume of lipemic human plasma required was calculated. The requiredvolume of lipemic plasma was added to a container. Sodium azide wasadded to 0.05%, and gentamicin was added to 0.05%. The solution wasstirred slowly at 2-8 degrees for 15 to 20 minutes, or until thepreservatives were completely dissolved. The solution was stored at 2-8degrees C., and used within 30 days. The material may be stored at −20degrees C. for up to one year.

EXAMPLE 5 Manufacture of Icteric Plasma (1× bilirubin, 0.03%)

The volume of 1× bilirubin, 0.03% (icteric plasma) was calculated usinga stock of 10× bilirubin 0.3%. Autoclaved DI water was added to acontainer. 1N NaOH was added in equal mass to the amount of 10×bilirubin, 0.3% that was subsequently added. A lid was placed on thecontainer, and the solution stirred slowly with an autoclaved stir barfor 30 minutes at 2-8 degrees C. An equal mass of NAT-DM EDTA (citrated)was added, and the solution mixed slowly for 15-20 minutes at 2-8degrees C. The pH was measured to determine that the pH ranged from 7.5to 8.0. The solution was stored at 2-8 degrees C.

EXAMPLE 6 Manufacture of Heparinized Human Plasma

Heparinized human plasma was added to a container. The volume was made0.05% sodium azide and 0.05% gentamicin sulfate. Caution was taken towear a dust mask when handling gentamicin sulfate. A lid was placed onthe container, and the solution stirred slowly at 2-8 degrees C. for15-20 minutes or until the preservatives were completely dissolved.Heparinized human plasma was stored at 2-8 C and used within 30 days ofpreparation. Alternatively, it may be stored at −20° C. for up to oneyear.

EXAMPLE 7 Preparation of Panel Members and Analyte Mixtures

Panel members were chosen according to the inhibitory substances to betested and at various concentrations. For example, an inhibition panelwas prepared for the following panel members: EDTA plasma (negativecontrol), haemolyzed blood (low concentration), haemolyzed blood (middleconcentration), haemolyzed blood (high concentration, heparinizedplasma, lipemic plasma, and icteric plasma (see Table 1).

The inhibition panel members were thawed at 37° C. for 15 to 20 minutes,vortexed vigorously to form homogenous suspensions of panel members andeliminate any precipitates that may form in panel members that containviscous substances such as haemoglobin or lipids. Any unused portionswere returned to the recommended storage conditions immediatelyfollowing use. Each panel member sample was provided in approximately 2mL vials.

TABLE 1 Interference Panel Members Panel Member Description 1 EDTAPlasma Negative 2 Haemolyzed Blood, Low 3 Haemolyzed Blood, Mid 4Haemolyzed Blood, High 5 Heparinized Plasma 6 Lipemic Plasma 7 IctericPlasma

The analyte of interest was then spiked into each panel member. Theanalyte of interest was in a physiological buffer, plasma, serum orother blood derivative that is compatible with the Inhibition Panelmembers disclosed herein. If the analyte of interest is a blood bornevirus such as HIV, HBV, HCV, HSV-1 or HSV-2, EBV or CMV appropriatelevels of the analytes may be purchased from a commercially availablesource (AcroMetrix Corporation).

The viral spike volume may be variable within the range of 1-50% of thetotal volume, depending on the viral load that was used for spiking. Forexample, if the total sample volume was 500 μL, the interference panelwas spiked with a viral stock ranging from 0.5 to 250 μL, for a finalvolume of 500 μL for individual members of the inhibition panel. Thedesired volume should not exceed more than 50% of the total volume, asthis may diminish the effective range of the potential interferingsubstance.

All interference panel members, including EDTA plasma, were spiked withan equal volume and copies of the desired analyte, so that theinterference/non-interference claims of the extraction and assay systemare properly verified.

In Table 2, HCV, the blood borne analyte of interest, was spiked intoeach panel member listed in Table 1. An equal volume of each panelmember (0.5 mL) was spiked with an equal volume (0.5 mL) of HCV to givea total volume of 1.0 mL for each sample. Also, an equal amount ofcopies of the target analyte were added using a known titer value of 5E5IU/mL, so that the interference/non-interference interpretation of theextraction and assay system were properly verified.

TABLE 2 Interference Panel Sample Preparation Volume of Total Volume ofpanel volume of viral spike member sample prep Panel Member (mL) (mL)(mL) EDTA Plasma 0.5 0.5 1.0 Haemolyzed 0.5 0.5 1.0 Plasma (low)Haemolyzed 0.5 0.5 1.0 Plasma (mid) Haemolyzed 0.5 0.5 1.0 Plasma (high)Heparinized Plasma 0.5 0.5 1.0 Lipemic Plasma 0.5 0.5 1.0 Icteric Plasma0.5 0.5 1.0

The analyte spike may be added directly to the inhibition panel membervials, or the inhibition panel members may be aliquoted into smallervolumes (additional vials not included) and then spiked with appropriatevolumes of the analyte. The analyte spike volume was variable within theflexible range of 1% to 50% of the total volume, depending on theconcentration that was used for spiking. For instance, if the totalvolume required for the initial extraction or assay procedure was 500μL, the analyte spike volume may range from 0.5 μL to 250 μL. Anappropriate volume of each member of the Inhibition Panel should then beadded to reach the final volume of 500 μL.

The desired analyte volume should not exceed more than 50% of the totalvolume, as this will diminish the effective range of the potentialinterfering substances. The inhibition panel may be used more than oncedepending on the input volume requirements of the particular extractionor assay methodology utilized.

EXAMPLE 8 Assay Evaluation

The panel members spiked with analyte of interest as shown in Tables 1and 2 were then utilized for the different extraction systems with 1-3replicas depending on the starting volume. Data in Table 3 shows theanalysis of HCV spiked into the inhibition panel members, extracted viaa commercial extraction system and analyzed within the reportabledynamic range of the assay (5E4 copies/mL) using a nucleic acidanalyzer, such as the COBAS TaqMan® 48 HCV ASR.

The level of interference caused by the challenging panel member wasquantified by comparison of either the delay in the threshold cycles(Ct's), or the decrease in the quantity of virus particles (using theEDTA plasma as optimal matrix) in the presence of the interferingsubstance.

Table 3 shows the inhibitory effect of Heparinized Plasma, LipimicPlasma and Icteric Plasma compared to EDTA plasma, which served asnon-inhibitory reference. The “Average Ct (cycle time)” was compared foreach panel member to EDTA plasma. A one Ct delay representedapproximately a 50% inhibition, meaning the titer could beunderestimated by a factor of two. A two cycle delay would indicate apossible four fold underestimation. The three haemolyzed plasmas showedpractically no inhibitory effect.

The inhibition panel can also be used to examine the function of QS(quantification standard), which is intended to monitor inhibition.Ideally, QS should be delayed by the same number of cycles as the targetCt. In this experiment it can be seen that a 7.9 cycle target Ct delaycaused by heparin is mimicked by a 7.3 cycle delay of the QS, whichstill allowed reporting of a reasonably accurate target titer. This kindof experiment was useful to demonstrate the robustness of an assay inthe presence of inhibitors.

TABLE 3 Performance of nucleic acid analyzer (COBAS TaqMan ®) 48 HCV ASRwith HCV spiked into Interference Panel Members Ct Average Bias Ct PanelCt (sam- Average bias Average Quantity Member (sample) ple) Ct (QS) (QS)Quantity Bias EDTA 29.7 0.0 33.0 0.0 3.16E+04 0 Plasma Haemol- 29.7 0.032.8 −0.2 2.76E+04 −4.00E+03 yzed Plasma (low) Haemol- 29.5 −0.2 32.7−0.3 2.91E+04 −2.53E+03 yzed Plasma (mid) Haemol- 29.8 0.1 33.0 0.02.74E+04 −4.20E+03 yzed Plasma (high) Heparin- 37.6 7.9 40.3 7.31.04E+04 −2.12E+04 ized Plasma Lipemic 32.6 2.9 35.1 2.1 1.99E+04−1.17E+04 Plasma Icteric 31.0 1.3 33.7 0.7 2.08E+04 −1.09E+04 Plasma

I claim:
 1. A method for determining the presence of substances in ananalytical assay comprising: adding a known amount of an analyte to twoor more members of an inhibition panel, wherein each member of theinhibition panel contains a potentially inhibition of the analyticalassay, performing the assay to measure the analyte, comparing ameasurement of reference plasma sample with the measurement of at leastone member of an inhibition panel, wherein the comparison indicates thepresence an actual inhibition of the analytical assay.
 2. The method ofclaim 1, wherein the comparison detects more than one substance thatinhibits the assay in the test sample.
 3. The method of claim 1, whereinthe analytical assay amplifies nucleic acids.
 4. The method of claim 3,wherein the analytical assay is PCR.
 5. The method of claim 1, whereinthe analyte is a nucleic acid.
 6. The method of claim 1, wherein theanalyte is an antibody.
 7. The method of claim 1, wherein the analyte isan antigen.
 8. The method of claim 1, wherein the test sample isextracted from serum.
 9. The method of claim 1, wherein the test sampleis extracted from plasma.
 10. The method of claim 1, wherein the testsample is extracted from plasma.
 11. The method of claim 1, wherein theinhibitory substance is selected from the group consisting ofhemoglobin, lipids, bilirubin, and heparin.
 12. A kit for evaluating theperformance of an analytical assay comprising: a panel comparing vialsof a plurality of inhibitory substances wherein each substance isquantified to a known concentration and volume, and analyte at a knownconcentration to be added to each panel member.
 13. The kit of claim 12,wherein the kit further comprises a reference sample free of inhibitorysubstance.
 14. The kit of claim 12, wherein an inhibitory substance isheparinzed.
 15. The kit of claim 12, wherein an inhibitory substance islipemic plasma.
 16. The kit of claim 12, wherein an inhibitory substanceis icteric plasma.
 17. The kit of claim 12, wherein an inhibitorysubstance is haemolytic blood.
 18. The kit of claim 12, wherein theanalyte is an antibody.
 19. The kit of claim 12, wherein the analyte isan antigen.
 20. The kit of claim 12, wherein the analyte is selectedfrom the group consisting of HIV, HBV, HCV, HSV-1, HSV-2, EBV or CMV.